l-Lactate treatment by photosynthetic cyanobacteria expressing heterogeneous l-lactate dehydrogenase

l-Lactate is a major waste compound in cultured animal cells. To develop a sustainable animal cell culture system, we aimed to study the consumption of l-lactate using a photosynthetic microorganism. As genes involved in l-lactate utilization were not found in most cyanobacteria and microalgae, we introduced the NAD-independent l-lactate dehydrogenase gene from Escherichia coli (lldD) into Synechococcus sp. PCC 7002. The lldD-expressing strain consumed l-lactate added to basal medium. This consumption was accelerated by expression of a lactate permease gene from E. coli (lldP) and an increase in culture temperature. Intracellular levels of acetyl-CoA, citrate, 2-oxoglutarate, succinate, and malate, and extracellular levels of 2-oxoglutarate, succinate, and malate, increased during l-lactate utilization, suggesting that the metabolic flux from l-lactate was distributed toward the tricarboxylic acid cycle. This study provides a perspective on l-lactate treatment by photosynthetic microorganisms, which would increase the feasibility of animal cell culture industries.

. Mouse C2C12 myoblasts and primary bovine myoblasts have been successfully cultured using cell extracts from Chlorella vulgaris, Chlorococcum littorale, and Arthrospira platensis as nutrients 6,7 . Thus, by treating waste compounds from animal cells (such as ammonium and l-lactate) using a photosynthetic microorganism, a sustainable animal cell culture system, that is, a circular cell culture (CCC) system, can be developed 8 (Fig. 1). Consumption of ammonium in the culture waste of C2C12 cells has already been achieved through the cultivation of C. vulgaris and C. littorale 9 . The potential application of the CCC system was previously shown using C. littorale, RL34 hepatocytes, and C2C12 myoblasts as producers of nutrients, growth factors, and muscles, respectively 8 . However, l-lactate consumption by photosynthetic microorganisms has not yet been investigated. l-Lactate is a major waste compound in cultured animal cells, and its accumulation in medium causes cytotoxic effects by changing the pH and osmolarity 10 . Therefore, l-lactate removal is a ubiquitous requirement in industries that utilize cultured animal cells and is necessary for optimal functioning of the CCC system. Cyanobacteria such as Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803 harbor d-lactate dehydrogenase (d-LDH; EC 1.1.1.28) as an enzyme involved in lactate metabolism and can produce d-lactate 11,12 (Fig. 2). The genetically engineered strains of PCC 6803 into which NAD-dependent l-lactate dehydrogenase (l-nLDH, EC 1.1.1.27) has been introduced can also produce l-lactate 13 . However, the ability of microalgae and cyanobacteria to consume l-/d-lactate remains unclear. Several heterotrophic bacteria, such as Escherichia coli, Corynebacterium glutamicum, and Pseudomonas aeruginosa, can utilize l-lactate with the help of NADindependent l-lactate dehydrogenase (l-iLDH, EC 1. 1 www.nature.com/scientificreports/ pyruvate [14][15][16] . For example, E. coli harbors the lactate operon, which is composed of the lldD (l-iLDH) and lldP (lactate permease) genes for l-lactate utilization as well as the lldR (regulatory protein) gene 14 .
The present study aimed to develop a method for l-lactate consumption using a photosynthetic microorganism. Most cyanobacteria and microalgae do not possess genes for l-lactate metabolism and cannot utilize l-lactate. Therefore, the ability for l-lactate utilization was added to PCC 7002 by heterogeneous expression of the lldD and lldP genes from E. coli. The distribution of metabolic flux derived from l-lactate was determined through metabolome analysis. This study provides a perspective on l-lactate treatment by photosynthetic microorganisms and establishes a method to develop cyanobacteria with the ability to utilize l-lactate. These findings will be valuable to industries using animal cell cultures and would increase the feasibility of the CCC system.

Results and discussion
l-Lactate utilization ability of cyanobacteria and microalgae in nature. l-Lactate has cytotoxic effects on animal cells 10 . In the present study, C2C12 cells were examined, and addition of l-lactate higher than 20 mM significantly decreased cell viability ( Supplementary Fig. 1). To achieve a sustainable CCC system 8 ( Fig. 1), we aimed to develop a method for l-lactate consumption using a photosynthetic microorganism. The conservation of genes involved in l-lactate metabolism was first investigated in silico to examine the l-lactate utilization ability of photosynthetic microorganisms in nature (Fig. 2). In this investigation, cyanobacteria and microalgae harboring l-iLDH (EC 1.1.2.3), l-lactate oxidase (LOX, EC 1.1.3.2), or lactate 2-monoxygenase (LMO, EC 1.13.12.4) genes were identified using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database 17 . l-iLDH and LOX convert l-lactate into pyruvate, and LMO converts l-lactate into acetate 18 . In contrast, l-nLDH (EC 1.1.1.27) synthesizes l-lactate from pyruvate reversibly or irreversibly 19 . Therefore, cyanobacteria and microalgae harboring LOX and LMO genes were not found. In addition, the l-iLDH gene is absent in most cyanobacteria and microalgae, except Aureococcus anophagefferens 20 , Crocosphaera watsonii 21 , Trichodesmium erythraeum 22 , and Rivularia sp. 23 , among which A. anophagefferens and T. erythraeum are known to cause harmful algal blooms. Thus, the present study found that l-lactate utilization genes are absent in most cyanobacteria and microalgae.
To experimentally examine the l-lactate utilization ability, several cyanobacteria and microalgae, i.e. Synechococcus sp. PCC 7002 24 , Anabaena sp. PCC 7120 25 , Arthrospira platensis NIES-39 26 , Chlamydomonas sp. KOR1 27 , and Pavlova sp. OPMS 30543 28 , were cultured phototrophically in the presence of l-lactate. The growth of these cyanobacteria and microalgae, except for PCC 7120, was not suppressed by 20 mM l-lactate, which is nearly equivalent to the culture waste of animal cells. However, lactate concentration in the medium did not change during cultivation ( Supplementary Fig. 2). These results suggest that few cyanobacteria and microalgae species in nature can utilize l-lactate. Therefore, genetic engineering should be a suitable approach for creating photosynthetic microorganisms capable of utilizing l-lactate.   www.nature.com/scientificreports/ Genetic engineering of cyanobacteria for l-lactate utilization. To develop photosynthetic microorganisms capable of l-lactate utilization, this study introduced heterogenous genes into the marine cyanobacterium PCC 7002, which is tolerant to salinity of animal medium. Because several heterotrophic bacteria can grow using l-lactate due to l-iLDH [14][15][16] , the present study employed l-iLDH encoded by the E. coli lldD gene. The lldP gene encoding lactate permease was previously shown to improve d-lactate export in cyanobacteria such as PCC 7002 and Synechococcus elongatus PCC 7942 12,29,30 . This study employed the lldP gene to enhance l-lactate uptake. The lldD and lldP genes were expressed in PCC 7002 cells using the constitutive trc promoter (Fig. 3A). These genetic elements were introduced into the ldhA gene site encoding d-LDH to prevent d-lactate synthesis from pyruvate by the native enzyme. Integration and complete segregation were confirmed using PCR ( Supplementary Fig. 3).
To evaluate the l-lactate utilization ability, PCC 7002 and the recombinant strains were phototrophically cultured in the presence of 20 mM l-lactate. During the early stage of cultivation, the recombinant strains showed more enhanced cell growth than PCC 7002 (Fig. 3B). l-lactate concentration in the medium significantly decreased in the lldD-expressing strain, while it remained unchanged in PCC 7002 (Fig. 3C). Additional introduction of the lldP gene enhanced l-lactate consumption by the lldD-expressing strain, and as a result, 20 mM (1.8 g·L −1 ) of l-lactate was completely consumed in 9 days. Thus, the l-lactate utilization ability was successfully added to PCC 7002 using the lldD/lldP genes. These results indicate that both LldD and LldP proteins were functional in cyanobacteria and that lldP can contribute to l-lactate import.
To accelerate l-lactate consumption, the culture temperature of the lldD/lldP-expressing strain was examined. During the initial 4 days, biomass concentration was not significantly different at 30-40 °C (Fig. 3D). In contrast, higher temperatures resulted in higher l-lactate consumption, and 20 mM l-lactate was completely consumed in 7 days at 35-40 °C (Fig. 3E). This result indicates that l-lactate utilization by the lldD/lldP-expressing strain can be enhanced by elevated temperatures probably because this temperature range is suitable for these E. coli enzymes. Thus, we established a method to develop cyanobacteria with the l-lactate utilization ability. Addition of a functional l-iLDH would also be valuable in adding l-lactate utilization ability to microalgae.

Distribution of metabolic flux from l-lactate. In lldD/lldP-expressing cells, pyruvate synthesized by
LldD is converted to other metabolites by intrinsic enzymes. To elucidate the distribution of the metabolic flux derived from l-lactate, metabolome analysis of lldD/lldP-expressing cells cultured in the absence and presence of l-lactate was performed. Lactate, pyruvate, and acetyl-CoA (AcCoA) accumulated in cells when supplemented with l-lactate (Fig. 4A). In addition, several metabolites of the tricarboxylic acid (TCA) cycle, that is, citrate, 2-oxoglutarate (2-OG), succinate, and malate, accumulated in the presence of l-lactate. These results indicate that the metabolic flux from l-lactate was largely distributed to the TCA cycle.
A significant intracellular accumulation of these metabolites should trigger their releases from lldD/lldPexpressing cells during l-lactate utilization. By analyzing the metabolites in the culture supernatant, we found that several metabolites increased extracellularly when supplemented with l-lactate (Fig. 4B). Extracellular levels of pyruvate, 2-OG, succinate, and malate in the lldD/lldP-expressing strain when supplemented with 20 mM l-lactate were 2,021.1, 496.7, 300.6, and 53.3 μM, respectively. The accumulation of intracellular metabolites by l-lactate utilization likely caused a significant release of these organic acids. A similar phenomenon, with elevated release of organic acids such as pyruvate and 2-OG, has been reported in glycogen-deficient cyanobacterial mutants [31][32][33] . Because 2-OG can be converted into glutamine, which is an essential and abundant amino acid in the medium for mammalian cell cultures 34 , the lldD/lldP-expressing strain may also be valuable for producing amino acids in animal cells.
Photosynthetic activity during l-lactate utilization. To examine whether the photosynthetic activity of lldD/lldP-expressing cells was affected by l-lactate utilization, the cells were analyzed in the presence and absence of l-lactate. First, O 2 evolution was analyzed on day 3 when the cells performed biomass production and l-lactate utilization (Fig. 3). The apparent O 2 evolution rate measured under light conditions significantly decreased in the presence of l-lactate (Fig. 5A). The O 2 consumption rate was measured in the dark and was found to be significantly increased when l-lactate was supplied to the medium. This might be due to the enhanced metabolic flux of the TCA cycle during l-lactate utilization (Fig. 4A) as the TCA cycle is the dominant source of NAD(P)H for respiratory electron transport 35 . The net photosynthetic O 2 evolution rate was calculated by subtracting the O 2 consumption rate from the apparent O 2 evolution rate and was found to be almost the same in the presence or absence of l-lactate.
To further examine photosynthetic activity, the CO 2 fixation activity of lldD/lldP-expressing cells was analyzed by in vivo 13 C labeling experiments. NaH 13 CO 3 was added to the medium on day 3 to supply the lldD/ lldP-expressing cells with 13 CO 2 . After incubation for 0-20 min, the 13 C fraction of metabolites in the Calvin-Benson-Bassham (CBB) cycle, which conducts photosynthetic CO 2 fixation by ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), was analyzed. The de novo synthesis of 3-phosphoglyceric acid (3-PGA), ribulose-5-phosphate (Ru5P), and ribulose-1,5-bisphosphate (RuBP) from CO 2 occurred even in the presence of l-lactate, although the 13 C fraction of 3-PGA was slightly decreased (Fig. 5B). These results reveal that photosynthesis in the lldD/lldP-expressing cells continued even during l-lactate utilization. In the presence of l-lactate, the de novo synthesis of Ru5P was not changed (Fig. 5B), while its accumulation significantly increased (Fig. 4A). These results are contrasting but not necessarily conflicting because these experiments analyzed for distinct metabolic parameters at different time points. The higher growth rate of l-lactate-assimilating cyanobacteria than that of PCC 7002 (Fig. 3B) might be due to the simultaneous utilization of CO 2 and l-lactate as carbon sources. Thus, this study established a method to consume l-lactate in animal cell cultures using photosynthetic www.nature.com/scientificreports/ cyanobacteria expressing the heterogenous lldD and lldP genes. The findings of this study will contribute to the development of a sustainable CCC system for the animal cell culture industry.   Construction of recombinant strains. The pUC118-based vectors, harboring the trc promoter and homology arms for the ldhA gene (SYNPCC7002_G0164) in the pAQ7 plasmid of Synechococcus sp., were used to introduce the lldD and lldP genes from E. coli via homologous recombination. The codon-optimized genes of LldD (NP_418062.1) and LldP (NP_418060.1) were prepared by the Genscript gene synthesis service and cloned into the vector using the In-Fusion HD Cloning Kit (Takara Bio USA, Inc., Mountain View, CA, USA). PCC 7002 was transformed as previously described 24 . Integration and complete segregation were confirmed by PCR using the specific primer pair 5′-AGA CAT TTC CCA CAG ACC ACA TCA AATTA-3′ and 5′-GGA TCA ATT TAC GTC TTT GTT GGC GCA-3′.

Measurement of lactate.
Each culture was centrifuged at 8000×g for 5 min. The supernatant was filtered using a Shim-pack SPR-Pb column (Shimadzu, Kyoto, Japan) and analyzed using a high-performance liquid chromatography system (Shimadzu) equipped with an Aminex HPX-87H column (Bio-Rad Laboratories, Hercules, CA, USA). l-Lactate (Sigma-Aldrich) was used as the quantitative standard to determine the lactate concentration using a calibration curve.

Metabolome analysis.
To prepare intracellular metabolites, a culture broth containing cells equivalent to 5 mg dry weight was mixed with four times the volume of 32.5% (v/v) methanol pre-cooled at − 30 °C. The mixture was centrifuged at 8000×g for 3 min at 4 °C. After complete removal of the supernatant, the cells were washed with 20 mM ammonium carbonate once and immediately resuspended in 1 mL of pre-cooled methanol containing 37.5 μM l-methionine sulfone and 37.5 μM piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES) as internal standards. The cell suspension (500 μL) was added to 200 μL ultrapure water and 500 μL chloroform pre-cooled at 4 °C and then vigorously mixed using vortexing for 30 s. After centrifugation at 14,000×g for 5 min at 4 °C, the aqueous layer was collected and filtered using an Amicon Ultra-0.5 Centrifugal Filter Unit UFC5003BK (Merck Millipore, Burlington, MA, USA) by centrifugation at 14,000×g at 4 °C. The sample (300 μL) was dried under vacuum using a centrifugal evaporator CEV-3100 (EYELA, Tokyo, Japan) and resuspended in 20 µL of ultrapure water. To prepare extracellular metabolites, the culture was centrifuged at 8000×g for 5 min. The supernatant (500 μL) was mixed with 500 μL chloroform pre-cooled at 4 °C by vortexing. After centrifugation at 14,000×g for 5 min at 4 °C, the upper layer was collected and filtered using UFC5003BK (Merck  www.nature.com/scientificreports/ Millipore) as described above. Next, 400 μM l-methionine sulfone and 400 μM PIPES were added as internal standards. The intracellular and extracellular samples were subjected to capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS) using a G7100 CE and G6224AA liquid chromatography-mass selective detector (LC/MSD) TOF system (Agilent Technologies, Santa Clara, CA, USA) 24 .
Analysis of photosynthesis. Cells were cultivated for 3 days under the conditions described above. To analyze the O 2 evolution rate, cultured cells were harvested, centrifuged at 8000×g for 5 min, and resuspended in fresh Medium A2 with or without 20 mM l-lactate to adjust the cell density to OD 750 = 5.0. The O 2 concentration in the cell suspension was measured using an oxygen electrode (Hansatech, King's Lynn, UK). During the measurements, the cell suspension was maintained at 30 °C and mixed using a magnetically controlled microstirrer.
The O 2 consumption rate of the cells was determined by measuring the O 2 concentration in the dark, while the apparent O 2 evolution rate was determined under illumination with a red LED light (200 μmol photons·m −2 ·s −1 ). The photosynthetic O 2 evolution rate was calculated by subtracting the O 2 consumption rate from the apparent O 2 evolution rate 24 .
To investigate carbon fixation from CO 2 , in vivo 13 C labeling of metabolites in the CBB cycle was performed. After 3 d of cultivation, the cell culture was added to 25 mM NaH 13 CO 3 as a carbon source and incubated under illumination with white fluorescent lamps (100 μmol photons·m −2 ·s −1 ). After labeling for 0-20 min, the intracellular metabolites were analyzed as described above 24 . The ratio of 13 C in the total carbon ( 13 C fraction) of the metabolite was determined based on the shifts between the 12 C and 13 C mass spectra.
Statistics and reproducibility. Data in this study are represented as mean ± standard deviation of three replicate experiments. Statistical significance was determined by Welch's t test.

Data availability
The data supporting the findings of this study are available from the corresponding author upon reasonable request.